1. Field of the Invention
This invention relates generally to the field of small conductance calcium activated potassium channels, and more specifically to two variants of the hSKCa3/SK3/KCNN3 gene used in the diagnosis, study, prevention and treatment of disorders related to these channels.
2. Background Information
Action potentials in vertebrate neurons are followed by an after hyperpolarization (AHP) that may persist for several seconds and may have profound consequences for the neuronal firing pattern. The AHP has several components, which are distinct and are mediated by different calcium activated potassium channels. Small conductance calcium activated potassium channels (SKCa, where “S” represents “small”) underlie slow components of the AHP, which are responsible for spike-frequency adaption (Hotson, J. R., and Prince, D. A., J. Neurophysiol. 43:409, 1980.).
Small conductance channels have a unitary conductance of 4–14 ps, are exquisitely sensitive to internal Ca2+, lack the property of voltage dependence and are blocked by nanomolar concentrations of the natural toxins apamin and scyllatoxin. These channels modulate the firing pattern of neurons via the generation of slow membrane afterhyperpolarizations (Nicoll, R. A., Science 241:545–551, 1988.). As small-conductance calcium-activated K+ channels are responsible for the afterhyperpolarization that follows each action potential in neurons, they thereby modulate neuronal firing frequency. Three phylogenetically related genes, KCNN1/SKCa1/SK1. KCNN2/SKCa2/SK2 and KCNN3/SKCa3/SK3 (Kohler et al., Science 273, 1709–1714, 1996), with a conserved gene structure (Ghanshani et al., J. Biol. Chem. 275, 37137–37149, 2000) encode this family of channels.
The sequence of the functional cDNA that encodes one subunit of the homotetrameric calcium-activated small-conductance K+ channel SKCa3/SK3/KCNN3 is set forth in U.S. Pat. No. 6,165,719. Additionally, the complete 160 kb sequence of the KCNN3 genomic locus has been reported. The sequence has been recorded and assigned accession number AF336797. It is known that within this structure KCNN3 has 8 exons (Sun et al., J. Hum. Genet. 46, 463–470, 2001.).
The SKCa are currents that have been described in a wide range of tissues, including brain (Lancaster, B. and Nicoll, R. A., J. Physiol. 389:187–203, 1987), peripheral neurons (Goh, J. W., and Pennefather, P. S., J. Physiol. 394:315–330, 1987), skeletal muscle (Romey, G., and Lazdunski, M., Biochem. Biophys. Res. Commun. 118:669–674, 1984) adrenal chromaffin cells (Neely, A., and Lingle, C. J., J. Physiol. 452:97–13 13, 1992), leukocytes (Grissmer, S., et al., J. Gen. Physiol. 99:63–84, 1992), erythrocytes (Hamill, O. P., J. Physiol. 319:97P–98P, 1981), colon (Lomax, R. B., et al., Gut 38:243–247, 1996), and airway epithelia (Welsh, M. J., and McCann, J. D., Proc. Natl. Acad. Sci. USA 82:8823–8826, 1985.). Certain types of SKCa channels have been distinguished by their sensitivities to the bee venom apamin, whereas other functionally related conductances appear insensitive (Sah, P., and AcLachlan, E. M., 1992, J. Neurophysiol. 74:1772–1776.). The distinguishing features of the SKCa channels from the maxi-K calcium activated (BK) potassium channels are the SKCa channels' low conductance (less than 50 pS), the weak or negligible dependence of their activity on membrane voltage, and their high affinity for calcium (EC50<1 μM) (e.g., Lancaster, B., and Zucker, R. S., J. Physiol. 475:229–239, 1994.).
Recently, Imbert et al. reported the cloning of six novel cDNAs, each containing one or more long CAG repeats. These cDNAs were isolated from a lymphoblastoid cell cDNA expression library generated from patients with autosomal dominant cerebellar ataxia with a monoclonal antibody specific for polyglutamine sequences (Imbert et al., Nature Genetics 14:285, 1996.). One of the cDNAs, designated AAD14, encoded the partial sequence of a putative protein of 228 amino acids in length. This polypeptide of unknown function contained two long polyglutamine stretches.
Kohler et al. first described a rat small conductance calcium activated potassium channel (rSKCa3) gene, and published the truncated form of the gene (Kohler et al., Science 274:1709, 1996.). An alignment of the known members of the small conductance calcium-activated potassium channels was described by Joiner et al. (Proc. Natl. Acad. Sci. 94: 11013, 1997), who cloned the hSK4 gene, using the potassium channel signature sequence TXXTXGYG (SEQ ID NO: 5). Ishii et al. (Proc. Natl. Acad. Sci. USA 94:11651–6, 1997) also cloned the intermediate conductance calcium-activated potassium channel h1K1, also known as hSK4.
Schizophrenia is a chronic disabling disorder with a lifetime morbid risk of 1% in the general population. The illness has a significant genetic component (Kendler, K. S., In: Relatives at Risk for Mental Disorders, Dunner, D. L., Gershon, E. S., and Barrett, J. E. (eds.), Raven Press, New York, pp. 247–263, 1988) and often develops in early adulthood. The disease is characterized by a constellation of symptoms including hallucinations and delusions, disordered thinking and concentration, inappropriate emotional responses, catatonia, erratic behavior, and social and occupational deterioration. Although still controversial, “anticipation” (an increase in severity through successive generations) has been found in studies of affected families (Bassett, A. S., and Honer, W. G., Am. J. Hum. Genet., 54:864–870, 1994.). Trinucleotide repeat expansions have been found to underlie several Mendelian hereditary neurological diseases (Ashley, C. T., Jr., and Warren, S. T., Annu. Rev. Genet., 29:703–728, 1995; Timchenko, L. T., and Caskey, C. T., FASEB J., 10:1589–1597, 1996; among others).
Several human hereditary neurological diseases, such as Huntington's disease, fragile X syndrome, myotonic dystrophy, spinal and bulbar muscular atrophy, Machado-Joseph disease, Friedrich's disease and spinocerebellar ataxia, are associated with expanded trinucleotide repeats (typically>35) within the coding region (e.g., Timchenko, L. T., and Caskey, C. T., FASEB J. 10: 1589–97, 1996; Hannan, A. J., J. Clin. Exp. Pharm. Physiol. 23:1015–20, 1996; Bates, G., Bioessays 18:1 75–8, 1996), untranslated sequences (e.g., Warren, S. T., and Ashley, C. T., Ann. Rev. Neurosci. 18:77–99, 1995; Tsilfidis, C., et al, Nature Genet. 1:192–195, 1992), or introns (Campuzano, V., et al., Science 271:1423–7, 1996) of genes. An association has been shown between the presence of anonymous CAG/CTG repeats and the development of schizophrenia and bipolar disease (O'Donovan, M. C., et al., Nature Genetics 10:380–1, 1995; O'Donovan et al., Psychological Med. 26:1145–1153, 1996; Cardino, A. G., et al., Brit. J. Psychiatry 169:766–771, 1996.).
KCNN3/SK3 has been mapped to chromosome 1q21 (Dror et al., Mol. Psych. 4, 254–260, 1999), a region strongly linked to schizophrenia (Brzustowicz et al., Science 288, 678–682, 2000) and more recently linked to Finnish Asperger syndrome, thought to be a childhood form of the disease.
KCNN3 contains two polymorphic polyglutamine repeats in the N-terminus. Such long repeats are highly over-represented in patients with schizophrenia compared to ethnic controls (Chandy et al., Mol. Psych. 3, 32–37, 1998; Dror et al., Mol. Psych. 4, 254–260, 1999.). Two independent studies have noted a strong association between long repeats and negative-symptom type schizophrenia (Cardno et al., Biol. Psych. 45, 1592–1596, 1999; Ritsner M, Modai I, Amir S, Halperin T, Weizman A, et al., An association of CAG repeats at the KCNN3 locus with symptom dimensions of schizophrenia. Biol Psychiatry, 2002. 51: 788–794).
Others have not been able to reproduce this association (Antonarakis et al., Am. J. Med. Genet. 88, 348–351, 1999, Joober et al, Am. J. Med. Genet. 88, 154–157, 1999; Bonner-Brilhault et al., Eur. J. Hum. Genet. 7, 247–250, 1999, Hawi et al., Mol. Psych. 4, 488–491, 1999; Chowdari et al., Mol. Psych. 5, 237–238, 2000.).
Intergenerational lengthening of expanded trinucleotide repeats is thought to underlie the phenomenon of “anticipation” (Ashley and Warren, 1995, supra) observed in schizophrenia and bipolar disease, wherein the disease worsens with subsequent generations (Johnson, J. E., et al., Amer. J. Med. Genet. 75:275–280, 1997; Lipp, O., et al., Psychiatric Genet. 5:S8, 1995; Serbanescu-Grigorow, M., et al., Psychiatric Genet. 5:S10, 1995.). The subset of these mutations caused by expansions of the CAG trinucleotide have been found only within the coding region of genes, and the encoded polyglutamine arrays may occur in different places within the open-reading-frame (Housman, D., Nature Genet. 10:3–4, 1995.). In the past few years, expanded “anonymous” CAG repeats in unidentified genes have been reported in patients with schizophrenia, with considerable overlap between allele distributions in patients and controls (O'Donovan, M. C., et al., Nature Genet. 10:380–381, 1995; Morris, A. G., et al., Hum. Mol. Genet., 4:1957–1961, 1995; O'Donovan, M. C., et al., Psychol. Med. 26:1145–1153, 1996; O'Donovan, M. C., and Owen, M. J., Psychol. Med. 26:1–6, 1996; O'Donovan, M. C., and Owen, M. J., Annals Med. 28:541–546, 1996a).
The expression of KCNN3/SKCa3 transcripts in dopaminergic neurons of the human midbrain have been demonstrated using in situ hybridization and quantitative PCR (Dror et al., Mol. Psych. 4, 254–260, 1999; Tomita et al, Mol Psych In press). Others have demonstrated that KCNN3/SKCa3 is the “pacemaker” that regulates neuronal firing frequency in dopaminergic neurons of the ventral tegmental area (Wolfart et al., J. Neurosci. 21, 3443–3456, 2001.).
The syndrome of schizophrenia has been long-associated with over-activity of the dopaminergic pathways (e.g. Farde et al., Schizophr. Res. 28, 157–162, 1997.). This finding has been pharmacologically re-confirmed by the fact that the drugs currently used to treat the disorder have a clinical potency linearly correlated with their affinity for the dopamine D2 receptor, a receptor most prominently displayed in the VTA and striatum. Apamin, a potent and selective inhibitor of SKCa channels, is known to block the post-spike afterhyperpolarization and change the firing pattern in dopaminergic neurons from a pacemaker-like discharge into a multiple bursting pattern (Shepard and Bunney Brain Res. 463, 380–384, 1988; Ping and Shepard Neuroreport 7, 809–814, 1996; Ping and Shepard J. Neurophysiol. 81, 977–984, 1999.). Bursting activity in turn has been associated with increased dopamine release (Steketee et al., J. Pharmacol. Exp Ther. 254, 711–719, 1990; Manley et al. J. Neurochem. 58, 1491–1498, 1992; Morikawa et al., J. Neurosci. 20, RC103 1–5, 2000.).
In one study, a 4 base-pair deletion in SKCa3 was reported in a patient with schizophrenia (Bowen et al., Mol. Psych. 6, 259–260, 2001), which truncates the protein at the N-terminus (referred to here as SKCa3-1/285). GFP-tagged SKCa3-1/285 localizes rapidly and exclusively to the nucleus of mammalian cells, whereas full-length SKCa3 is excluded from the nucleus and expresses as functional channels (Miller et al., J. Biol. Chem. 276, 27753–27756, 2001.). Such neuronal nuclear inclusions have been implicated in apoptosis in other trinucleotide repeat diseases, such as Huntington disease. Postmortem and neuroimaging studies have resulted in observations that there may be an alteration in the neuronal architecture in schizophrenia (McGlashan and Hoffman, Arch. Gen. Psych. 57, 637–648, 2000; Halliday, Clin. Exp. Pharmacol. Physiol. 28, 64–654, 2001.).
SKCa3-1/285 dominant-negatively suppresses endogenous SKCa2 currents in Jurkat T cells. While the mechanism of suppression remains unknown, it is hypothesized that the suppression occurs via co-assembly into non-functional tetramers (Miller et al., J. Biol. Chem. 276, 27753–27756, 2001.). Dominant-negative suppression by SKCa3–1/285 could lead to a global reduction of the entire family of SKCa1–SKCa3 channels in a fashion more potent than traditional allele-specific genetic suppression involving a single locus. Such reduction of all endogenous SKCa currents, many of them in dopaminergic neurons, might have effects analogous to the SKCa blocker apamin.